(a) Field of the Invention
The invention relates to a method for increasing the growth of plant cell culture for the production of economically important complex chemicals of plant origin (phytochemicals) at an industrial level.
(b) Description of Prior Art
Phytochemicals are non-proteinic biomolecules which cannot be synthesized at reasonable yields and costs by conventional chemical processes nor can they be produced through genetic manipulation of microorganisms due to the complex, and often poorly understood, biochemical pathways involved. The production of these precious molecules is mostly achieved through the extraction and purification, at low yields (&lt;1-5%), of imported exotic plant biomass, whose reproductive agriculture and secure long term supply are often very difficult, if not impossible to guarantee. Consequently, these supply problems have seriously hindered the development of these unique biomolecules into valuable active principles for the pharmaceutical, nutraceutical and cosmetic industries.
The culture of plant cells has been explored since the 1960's as a viable alternative for the production of complex phytochemicals (secondary metabolites) of industrial interest. However, this research which included the selection of better performing cell lines and the development of specific growth and production media, and of immobilized, organ and transformed cultures, resulted in no true success in achieving economical productivity levels. Most of these studies were performed using small scale, uncontrolled and unmonitored solid and liquid flask cultures and yielded generally unreproducible low production levels (&lt;100-200 mg L.sup.-1 in 14-28 days). Furthermore, secondary metabolites are mostly retained intracellularly and genetic manipulations to improve production have not been successful.
Nevertheless, a certain number of valuable advances were achieved over the years. Productive normal and transformed plant cell lines and production protocols were developed for a few secondary metabolites of industrial interest. Properly configured recombinant proteins and antibodies have been cloned and produced in plants and cultured plant cells. Using conventional bioreactors, plant cells can be cultivated at large scale (20,000-75,000 L) to compensate for the low volumetric productivities achieved, but always with lower phytochemical production than obtained in flasks.
Consequently, plant cell based bioprocesses for the production of valuable phytochemicals remain presently uneconomical due to the low productivities of the basic culture process and to the high investments in the large bioreactor systems required to compensate for their low production rate. This type of bioprocess comprises basically three stages: 1) a first stage where the plant cell biomass is grown to produce a high concentration; 2) a production stage during which this biomass is stimulated or challenged to biosynthesize the secondary metabolites of interest at high rate and concentration; and 3) a final stage of extraction and purification of phytochemicals from the culture broth. This last stage (downstream processing) is performed using conventional chemical engineering technologies.
Most research in this field has been focused, with some success, on improving the second, more glamorous stage of this bioprocess, i.e. developing culture methods (production media, transformed and organ cultures, elicitation, genetic manipulation etc.) to induce secondary metabolism in, and to maximize phytochemicals production by the plant cell biomass. The first stage of this bioprocess, a key issue with respect to secondary metabolite productivity, has rarely been studied in depth. In all cases, high concentrations (.about.30-50.sup.+ g dry biomass L.sup.-1) of productive biomass were achieved using high sugar concentrations. The biomass growth of these cultures under conventional, static (batch) conditions is slow (division time .about.24-72 h) but can attain high wet biomass concentrations (&gt;300 g L.sup.-1).
However, the basic effective growth behavior of plant cells cultivated in vitro consists of two distinct phases: cell division (which is indicative of cellular proliferation) followed by cell expansion (biomass growth).
Sargent et al. have investigated the conditioning effects of nutrient medium on only biomass growth. However, biomass growth is not indicative of cellular proliferation and vice versa. In fact, at the end of the cellular growth phase, the biomass continue to increase due to an uptake of water, carbohydrates, nitrate, and other macronutrients.
Similarly, Mori et al. is only concerned with the biomass concentration, which cannot be directly correlated to cellular growth.
In the field of plant cell culture, no group has ever clearly characterized, let alone measured both phases in culture. Only the increase of biomass concentration is usually measured to quantify growth. According to our work (Pepin, M. F. et al. (1995) Biotechnology and Bioengineering, 47:131-138), under normal (batch) growth conditions not limited by the availability of carbohydrates and dissolved oxygen, the division of cultured plant cell stops after the first 3 to 7 days of the typical 14-21 day duration of the biomass growth phase. This gives rise to a characteristic respiration pattern of the culture, plateauing at the end of cell division.
Thereafter, culture growth occurs only by cell and biomass expansion upon the uptake of water, carbohydrates, nitrate, and other macronutrients. This phenomenon was observed for three different plant cell species, Vitis vinifera (Pepin, M. F. et al. (1995) Biotechnology and Bioengineering, 47:131-138), Eschscholtzia californica and Ginkgo biloba, which indicates that it characterizes the growth behavior of many, if not all, plant cells cultured in vitro.
In this context, it would be highly desirable to be provided with a novel culture method to improve the cellular growth of in vitro cultivated plant cells in order to lower the duration of the first (growth) stage and maximize the cell concentration of plant cell based bioprocesses. This culture method could then be combined with other culture techniques developed to induce secondary metabolites as well as recombinant proteins and antibodies production in order that very high, economical productivity levels may be obtained from plant cell based bioprocesses.